Novel human endothelin converting enzyme-like proteins and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

The present application claims priority to U.S. Provisional ApplicationsNos. 60/156,102 and 60/176,689 which were filed on Sep. 24, 1999 andJan. 18, 2000, respectively, and are herein incorporated by reference intheir entirety.

1. INTRODUCTION

The present invention relates to the discovery, identification, andcharacterization of novel human polynucleotides encoding proteins thatshare sequence similarity with mammalian endothelin converting enzymes.The invention encompasses the described polynucleotides, host cellexpression systems, the encoded proteins, fusion proteins, polypeptidesand peptides, antibodies to the encoded proteins and peptides, andgenetically engineered animals that either lack or over express thedisclosed genes, antagonists and agonists of the proteins, and othercompounds that modulate the expression or activity of the proteinsencoded by the disclosed genes that can be used for diagnosis, drugscreening, clinical trial monitoring, and the treatment of physiologicaldisorders.

2. BACKGROUND OF THE INVENTION

Endothelin converting enzymes cleave endothelin precursor protein to itsbiologically active product. Given the strong vasoconstrictive activityof endothelins and their importance in, for example, renal andcardiovascular pathogenesis, methods of modulating endothelin productionand activity have been subject to significant scientific scrutiny.

3. SUMMARY OF THE INVENTION

The present invention relates to the discovery, identification, andcharacterization of nucleotides that encode novel human proteins, andthe corresponding amino acid sequences of these proteins. The novelhuman proteins (NHPs) novel human proteins, and the corresponding aminoacid sequences of these proteins. The novel human proteins (NHPs)described for the first time herein share structural similarity withanimal endothelin converting enzymes. As such, the NHPs may be involvedin regulating (i.e., directly or indirectly activating or inhibiting)endothelin activity in human cells and/or tissues. The described NHPsrepresent a new protein having a range of homologs and orthologs from avariety of species and phyla.

The novel human nucleic acid sequences described herein, encodeproteins/open reading frames (ORF) of 255 and 883 amino acids in length(see SEQ ID NOS: 2 and 4).

The invention also encompasses agonists and antagonists of the describedNHPs, including small molecules, large molecules, mutant NHPs, orportions thereof that compete with native NHPs, NHP peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHPs (e.g., antisense and ribozymemolecules, and gene or regulatory sequence replacement constructs) or toenhance the expression of the described NHP polynucleotides (e.g.,expression constructs that place the described gene under the control ofa strong promoter system), and transgenic animals that express a NHPtransgene, or “knock-outs” (which can be conditional) that do notexpress a functional NHP.

Further, the present invention also relates to processes for identifyingcompounds that modulate, i.e., act as agonists or antagonists, of NHPexpression and/or NHP product activity that utilize purifiedpreparations of the described NHP and/or NHP product, or cellsexpressing the same. Such compounds can be used as therapeutic agentsfor the treatment of any of a wide variety of symptoms associated withbiological disorders or imbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

The Sequence Listing provides the sequences of the described endothelinconverting enzyme-like ORFs that encode the described NHP amino acidsequences. SEQ ID NO: 5 describes the a NHP ORF (SEQ ID NO:1) withflanking sequences.

5. DETAILED DESCRIPTION OF THE INVENTION

The NHPs, described for the first time herein, are novel proteins thatare expressed in, inter alia, human cell lines, and fetal brain,cerebellum, thymus, spleen, lymph node, bone marrow, trachea, kidney,liver, prostate, testis, thyroid, adrenal gland, pancreas, salivarygland, stomach, small intestine, colon, muscle, adipose, esophagus,bladder, cervix, rectum, and pericardium cells. The described sequenceswere compiled from gene trapped cDNAs and clones isolated from a humanliver cDNA library (Edge Biosystems, Gaithersburg, Md.), as well aspublished sequences that did not represent or identify regions of thepresently described proteins. Given the important physiological role ofendothelin, endothelin converting enzymes have been subject toconsiderable scrutiny as described in U.S. Pat. Nos. 5,736,376,5,688,640 (describing recombinant expression and screening assays),5,338,726 (describing inhibitors), and 5,462,869 (describing generalmethods of purifying such proteins), all of which are herebyincorporated by reference in their entirety.

The present invention encompasses the nucleotides presented in theSequence Listing, host cells expressing such nucleotides, the expressionproducts of such nucleotides, and: (a) nucleotides that encode mammalianhomologs of the described genes, including the specifically describedNHP, and the NHP related products; (b) nucleotides that encode one ormore portions of a NHP that correspond to functional domains, and thepolypeptide products specified by such nucleotide sequences, includingbut not limited to the novel regions of any active domain(s); (c)isolated nucleotides that encode mutant versions, engineered ornaturally occurring, of a NHP in which all or a part of at least onedomain is deleted or altered, and the polypeptide products specified bysuch nucleotide sequences, including but not limited to soluble proteinsand peptides in which all or a portion of the signal sequence isdeleted; (d) nucleotides that encode chimeric fusion proteins containingall or a portion of a coding region of a NHP, or one of its domains(e.g., a receptor binding domain, accessory protein/self-associationdomain, etc.) fused to another peptide or polypeptide; or (e)therapeutic or diagnostic derivatives of the described polynucleotidessuch as oligonucleotides, antisense polynucleotides, ribozymes, dsRNA,or gene therapy constructs comprising a sequence first disclosed in theSequence Listing.

As discussed above, the present invention includes: (a) the human DNAsequences presented in the Sequence Listing (and vectors comprising thesame) and additionally contemplates any nucleotide sequence encoding acontiguous NHP open reading frame (ORF) that hybridizes to a complementof a DNA sequence presented in the Sequence Listing under highlystringent conditions, e.g., hybridization to filter-bound DNA in 0.5 MNaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M. et al., eds., 1989,Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3)and encodes a functionally equivalent gene product. Additionallycontemplated are any nucleotide sequences that hybridize to thecomplement of the DNA sequence that encode and express an amino acidsequence presented in the Sequence Listing under moderately stringentconditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al.,1989, supra), yet still encode a functionally equivalent NHP product.Functional equivalents of a NHP include naturally occurring NHPs presentin other species and mutant NHPs whether naturally occurring orengineered (by site directed mutagenesis, gene shuffling, directedevolution as described in, for example, U.S. Pat. No. 5,837,458, hereinincorporated by reference). The invention also includes degeneratenucleic acid variants of the disclosed NHP polynucleotide sequences.

Additionally contemplated are polynucleotides encoding NHP ORFs, ortheir functional equivalents, encoded by polynucleotide sequences thatare about 99, 95, 90, or about 85 percent similar or identical tocorresponding regions of SEQ ID NO:1 (as measured by BLAST sequencecomparison analysis using, for example, the GCG sequence analysispackage using standard default settings).

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of, thedescribed NHP gene nucleotide sequences. Such hybridization conditionsmay be highly stringent or less highly stringent, as described above. Ininstances where the nucleic acid molecules are deoxyoligonucleotides(“DNA oligos”), such molecules are generally about 16 to about 100 baseslong, or about 20 to about 80, or about 34 to about 45 bases long, orany variation or combination of sizes represented therein thatincorporate a contiguous region of sequence first disclosed in theSequence Listing. Such oligonucleotides can be used in conjunction withthe polymerase chain reaction (PCR) to screen libraries, isolate clones,and prepare cloning and sequencing templates, etc.

Alternatively, such NHP oligonucleotides can be used as hybridizationprobes for screening libraries, and assessing gene expression patterns(particularly using a micro array or high-throughput “chip” format).Additionally, a series of the described NHP oligonucleotide sequences,or the complements thereof, can be used to represent all or a portion ofthe described NHP sequences. The oligonucleotides, typically betweenabout 16 to about 40 (or any whole number within the stated range)nucleotides in length may partially overlap each other and/or the NHPsequence may be represented using oligonucleotides that do not overlap.Accordingly, the described NHP polynucleotide sequences shall typicallycomprise at least about two or three distinct oligonucleotide sequencesof at least about 18, and preferably about 25, nucleotides in lengththat are each first disclosed in the described Sequence Listing. Sucholigonucleotide sequences may begin at any nucleotide present within asequence in the Sequence Listing and proceed in either a sense(5′-to-3′) orientation vis-a-vis the described sequence or in anantisense orientation.

For oligonucleotide probes, highly stringent conditions may refer, e.g.,to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-baseoligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and60° C. (for 23-base oligos). These nucleic acid molecules may encode oract as NHP gene antisense molecules, useful, for example, in NHP generegulation (for and/or as antisense primers in amplification reactionsof NHP gene nucleic acid sequences). With respect to NHP generegulation, such techniques can be used to regulate biologicalfunctions. Further, such sequences may be used as part of ribozymeand/or triple helix sequences that are also useful for NHP generegulation.

Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety which isselected from the group including but not limited to 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide can also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide will compriseat least one modified phosphate backbone selected from the groupconsisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHP.

Oligonucleotides of the invention can be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides can be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

Low stringency conditions are well known to those of skill in the art,and will vary predictably depending on the specific organisms from whichthe library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual (and periodic updates thereof),Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, N.Y.

Alternatively, suitably labeled NHP nucleotide probes can be used toscreen a human genomic library using appropriately stringent conditionsor by PCR. The identification and characterization of human genomicclones is helpful for identifying polymorphisms (including, but notlimited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

Further, a NHP gene homolog can be isolated from nucleic acid from anorganism of interest by performing PCR using two degenerate or “wobble”oligonucleotide primer pools designed on the basis of amino acidsequences present within the NHP product disclosed herein. The templatefor the reaction may be total RNA, mRNA, and/or cDNA obtained by reversetranscription of mRNA prepared from, for example, human or non-humancell lines or tissue, such as prostate, rectum, colon, or adrenal gland,known or suspected to express an allele of a NHP gene. The PCR productcan be subcloned and sequenced to ensure that the amplified sequencesrepresent the sequence of the desired NHP gene. The PCR fragment canthen be used to isolate a full length cDNA clone by a variety ofmethods. For example, the amplified fragment can be labeled and used toscreen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

PCR technology can also be used to isolate full length cDNA sequences.For example, RNA can be isolated, following standard procedures, from anappropriate cellular or tissue source (i.e., one known, or suspected, toexpress a NHP gene). A reverse transcription (RT) reaction can beperformed on the RNA using an oligonucleotide primer specific for themost 5′ end of the amplified fragment for the priming of first strandsynthesis. The resulting RNA/DNA hybrid may then be “tailed” using astandard terminal transferase reaction, the hybrid may be digested withRNase H, and second strand synthesis may then be primed with acomplementary primer. Thus, cDNA sequences upstream of the amplifiedfragment can be isolated. For a review of cloning strategies that can beused, see e.g., Sambrook et al., 1989, supra.

A cDNA encoding a mutant NHP gene can be isolated, for example, by usingPCR. In this case, the first cDNA strand may be synthesized byhybridizing an oligo-dT oligonucleotide to mRNA isolated from tissueknown or suspected to be expressed in an individual putatively carryinga mutant NHP allele, and by extending the new strand with reversetranscriptase. The second strand of the cDNA is then synthesized usingan oligonucleotide that hybridizes specifically to the 5′ end of thenormal gene. Using these two primers, the product is then amplified viaPCR, optionally cloned into a suitable vector, and subjected to DNAsequence analysis through methods well known to those of skill in theart. By comparing the DNA sequence of the mutant NHP allele to that of acorresponding normal NHP allele, the mutation(s) responsible for theloss or alteration of function of the mutant NHP gene product can beascertained.

Alternatively, a genomic library can be constructed using DNA obtainedfrom an individual suspected of or known to carry a mutant NHP allele(e.g., a person manifesting a NHP-associated phenotype such as, forexample, obesity, high blood pressure, etc.), or a cDNA library can beconstructed using RNA from a tissue known, or suspected, to express amutant NHP allele. A normal NHP gene, or any suitable fragment thereof,can then be labeled and used as a probe to identify the correspondingmutant NHP allele in such libraries. Clones containing mutant NHP genesequences can then be purified and subjected to sequence analysisaccording to methods well known to those skilled in the art.

Additionally, an expression library can be constructed utilizing cDNAsynthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant NHP allele in an individual suspected ofor known to carry such a mutant allele. In this manner, gene productsmade by the putatively mutant tissue may be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against a normal NHP product, as described below. (For screeningtechniques, see, for example, Harlow, E. and Lane, eds., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Press, Cold SpringHarbor.)

Additionally, screening can be accomplished by screening with labeledNHP fusion proteins, such as, for example, alkaline phosphatase-NHP orNHP-alkaline phosphatase fusion proteins. In cases where a NHP mutationresults in an expressed gene product with altered function (e.g., as aresult of a missense or a frameshift mutation), polyclonal antibodies toa NHP are likely to cross-react with a corresponding mutant NHP geneproduct. Library clones detected via their reaction with such labeledantibodies can be purified and subjected to sequence analysis accordingto methods well known in the art.

The invention also encompasses (a) DNA vectors that contain any of theforegoing NHP coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHP coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example, baculovirus as described in U.S. Pat. No. No. 5,869,336 herein incorporated byreference); (c) genetically engineered host cells that contain any ofthe foregoing NHP coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHP gene under the control of an exogenously introducedregulatory element (i.e., gene activation). As used herein, regulatoryelements include but are not limited to inducible and non-induciblepromoters, enhancers, operators and other elements known to thoseskilled in the art that drive and regulate expression. Such regulatoryelements include but are not limited to the cytomegalovirus hCMVimmediate early gene, regulatable, viral (particularly retroviral LTRpromoters) the early or late promoters of SV40 adenovirus, the lacsystem, the trp system, the TAC system, the TRC system, the majoroperator and promoter regions of phage lambda, the control regions of fdcoat protein, the promoter for 3-phosphoglycerate kinase (PGK), thepromoters of acid phosphatase, and the promoters of the yeast α-matingfactors.

The present invention also encompasses antibodies and anti-idiotypicantibodies (including Fab fragments), antagonists and agonists of theNHP, as well as compounds or nucleotide constructs that inhibitexpression of a NHP gene (transcription factor inhibitors, antisense andribozyme molecules, or gene or regulatory sequence replacementconstructs), or promote the expression of a NHP (e.g., expressionconstructs in which NHP coding sequences are operatively associated withexpression control elements such as promoters, promoter/enhancers,etc.).

The described NHP or NHP peptides, NHP fusion proteins, NHP nucleotidesequences, antibodies, antagonists and agonists can be used in thedetection of mutant NHPs or inappropriately expressed NHP for thediagnosis of disease. The NHP or NHP peptides, NHP fusion proteins, NHPnucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canalso be used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of NHP inthe body. The use of engineered host cells and/or animals can offer anadvantage in that such systems allow not only for the identification ofcompounds that bind to the endogenous receptor for an NHP, but can alsoidentify compounds that trigger NHP-mediated pathways.

Finally, the NHP products can be used as therapeutics. For example,soluble derivatives such as NHP peptides/domains corresponding to theNHPs, NHP fusion protein products (especially NHP-Ig fusion proteins,i.e., fusions of a NHP, or a domain of a NHP, to an IgFc), NHPantibodies and anti-idiotypic antibodies (including Fab fragments),antagonists or agonists (including compounds that modulate signaltransduction which may act on downstream targets in a NHP-mediatedpathway) can be used to directly treat diseases or disorders. Forinstance, the administration of an effective amount of soluble NHP, or aNHP-IgFc fusion protein or an anti-idiotypic antibody (or its Fab) thatmimics the NHP could activate or effectively antagonize the endogenousNHP receptor. Nucleotide constructs encoding such NHP products can beused to genetically engineer host cells to express such products invivo; these genetically engineered cells function as “bioreactors” inthe body delivering a continuous supply of a NHP, a NHP peptide, or aNHP fusion protein to the body. Nucleotide constructs encodingfunctional NHP, mutant NHPs, as well as antisense and ribozyme moleculescan also be used in “gene therapy” approaches for the modulation of NHPexpression. Thus, the invention also encompasses pharmaceuticalformulations and methods for treating biological disorders.

Various aspects of the invention are described in greater detail in thesubsections below.

5.1 The NHP Sequences

The cDNA sequences (SEQ ID NOS: 1 and 3) and the corresponding deducedamino acid sequence (SEQ ID NOS: 2 and 4) of the described NHPs arepresented in the Sequence Listing. The NHP genes were obtained from ahuman liver cDNA library using probes and/or primers generated fromhuman gene trapped sequence tags. Expression analysis has providedevidence that the described NHPs can be expressed in a wide range ofhuman tissues as well as gene trapped human cells.

SEQ ID NO:3 describes a full length ORF with flanking 5′ and 3′sequences.

5.2 NHP Polypeptides

The described NHPs, NHP polypeptides, NHP peptide fragments, mutated,truncated, or deleted forms of the NHPs, and/or NHP fusion proteins canbe prepared for a variety of uses. These uses include but are notlimited to the generation of antibodies, as reagents in diagnosticassays, for the identification of other cellular gene products relatedto a NHP, as reagents in assays for screening for compounds that can beas pharmaceutical reagents useful in the therapeutic treatment ofmental, biological, or medical disorders and disease.

The Sequence Listing discloses the amino acid sequence encoded by thedescribed NHP genes. The NHPs each display a initiator methionine in aDNA sequence context consistent with a translation initiation sites, andhave structural features characteristic of related endothelin convertingproteins.

The NHP amino acid sequences of the invention include the amino acidsequences presented in the Sequence Listing as well as analogues andderivatives thereof. Further, corresponding NHP homologues from otherspecies are encompassed by the invention. In fact, any NHP proteinencoded by the NHP nucleotide sequences described above are within thescope of the invention, as are any novel polynucleotide sequencesencoding all or any novel portion of an amino acid sequence presented inthe Sequence Listing. The degenerate nature of the genetic code is wellknown, and, accordingly, each amino acid presented in the SequenceListing, is generically representative of the well known nucleic acid“triplet” codon, or in many cases codons, that can encode the aminoacid. As such, as contemplated herein, the amino acid sequencespresented in the Sequence Listing, when taken together with the geneticcode (see, for example, Table 4-1 at page 109 of “Molecular CellBiology”, 1986, J. Darnell et al. eds., Scientific American Books, NewYork, N.Y., herein incorporated by reference) are genericallyrepresentative of all the various permutations and combinations ofnucleic acid sequences that can encode such amino acid sequences.

The invention also encompasses proteins that are functionally equivalentto a NHP encoded by the presently described nucleotide sequences asjudged by any of a number of criteria, including, but not limited to,the ability to bind and cleave a substrate of a NHP, or the ability toeffect an identical or complementary downstream pathway, or a change incellular metabolism (e.g., proteolytic activity, ion flux, tyrosinephosphorylation, etc.). Such functionally equivalent NHP proteinsinclude, but are not limited to, additions or substitutions of aminoacid residues within the amino acid sequence encoded by the NHPnucleotide sequences described above, but which result in a silentchange, thus producing a functionally equivalent gene product. Aminoacid substitutions can be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

A variety of host-expression vector systems can be used to express theNHP nucleotide sequences of the invention. Such expression systems alsoencompass engineered host cells that express a NHP, or functionalequivalent, in situ. Purification or enrichment of a NHP from suchexpression systems can be accomplished using appropriate detergents andlipid micelles and methods well known to those skilled in the art.However, such engineered host cells themselves may be used in situationswhere it is important not only to retain the structural and functionalcharacteristics of the NHP, but to assess biological activity, e.g., indrug screening assays. Where, as in the present instance, the NHPpeptide or polypeptide is thought to be a membrane protein, expressionsystems can be engineered that produce soluble derivatives of a NHP(corresponding to a NHP extracellular and/or intracellular domains, ortruncated polypeptides lacking one or more transmembrane domains) and/orNHP fusion protein products (especially NHP-Ig fusion proteins, i.e.,fusions of a NHP domain, e.g., ECD, ΔTM to an IgFc), NHP antibodies andanti-idiotypic antibodies (including Fab fragments) which can be used intherapeutic applications. Preferably, the above expression systems areengineered to allow the desired peptide or polypeptide to be recoveredfrom the culture media.

The expression systems that can be used for purposes of the inventioninclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing NHP nucleotidesequences; yeast ( e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing NHP nucleotidesequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing NHP sequences; plantcell systems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing NHP nucleotide sequences; or mammalian cell systems(e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells. (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHPproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing NHP, or for raising antibodies to a NHP,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited, to the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequencemay be ligated individually into the vector in frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouye& Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may alsobe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The PGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

In an insect system, Autographa californica nuclear polyhidrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. A NHP gene coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). Successful insertion of NHP genecoding sequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed (e.g., see Smith et al., 1983, J. Virol. 46:584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the NHP nucleotide sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing a NHP product in infected hosts (e.g., See Logan & Shenk,1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiationsignals may also be required for efficient translation of inserted NHPnucleotide sequences. These signals include the ATG initiation codon andadjacent sequences. In cases where an entire NHP gene or cDNA, includingits own initiation codon and adjacent sequences, is inserted into theappropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only a portion of a NHPcoding sequence is inserted, exogenous translational control signals,including, perhaps, the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (SeeBittner et al., 1987, Methods in Enzymol. 153:516-544).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3, WI38, and in particular, human cell lines.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe NHP sequences described above can be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells can beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the NHPproduct. Such engineered cell lines can be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the NHP product.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

Alternatively, any fusion protein can be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA88:8972-8976). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺.nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

5.3 Antibodies to NHP Products

Antibodies that specifically recognize one or more epitopes of a NHP, orepitopes of conserved variants of a NHP, or peptide fragments of a NHPare also encompassed by the invention. Such antibodies include but arenot limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

The antibodies of the invention may be used, for example, in thedetection of NHP in a biological sample and may, therefore, be utilizedas part of a diagnostic or prognostic technique whereby patients may betested for abnormal amounts of NHP. Such antibodies may also be utilizedin conjunction with, for example, compound screening schemes for theevaluation of the effect of test compounds on expression and/or activityof a NHP gene product. Additionally, such antibodies can be used inconjunction gene therapy to, for example, evaluate the normal and/orengineered NHP-expressing cells prior to their introduction into thepatient. Such antibodies may additionally be used as a method for theinhibition of abnormal NHP activity. Thus, such antibodies may,therefore, be utilized as part of treatment methods.

For the production of antibodies, various host animals may be immunizedby injection with a NHP, a NHP peptide (e.g., one corresponding to afunctional domain of a NHP), truncated NHP polypeptides (NHP in whichone or more domains have been deleted), functional equivalents of theNHP or mutated variant of the NHP. Such host animals may include but arenot limited to pigs, rabbits, mice, goats, and rats, to name but a few.Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to Freund'sadjuvant (complete and incomplete), mineral salts such as aluminumhydroxide or aluminum phosphate, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and Corynebacterium parvum. Alternatively, the immune response could beenhanced by combination and or coupling with molecules such as keyholelimpet hemocyanin, tetanus toxoid, diptheria toxoid, ovalbumin, choleratoxoid or fragments thereof. Polyclonal antibodies are heterogeneouspopulations of antibody molecules derived from the sera of the immunizedanimals.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Such technologies are described in U.S. Pat. Nos. 6,075,181 and5,877,397 and their respective disclosures which are herein incorporatedby reference in their entirety.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) can be adapted to produce single chainantibodies against NHP gene products. Single chain antibodies are formedby linking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, such fragments include, but are notlimited to: the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibodies to a NHP can, in turn, be utilized to generate anti-idiotypeantibodies that “mimic” a given NHP, using techniques well known tothose skilled in the art. (See, e.g., Greenspan & Bona, 1993, FASEB J7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438). Forexample antibodies which bind to a NHP domain and competitively inhibitthe binding of NHP to its cognate receptor can be used to generateanti-idiotypes that “mimic” the NHP and, therefore, bind and activate orneutralize a receptor. Such anti-idiotypic antibodies or fragments ofsuch anti-idiotypes can be used in therapeutic regimens involving a NHPsignaling pathway.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims. All patents, patentapplications, and publications cited herein are incorporated byreference in their entirety.

1-6. (canceled)
 7. An isolated nucleic acid molecule that encodes theamino acid sequence shown in SEQ ID NO:4.
 8. The isolated nucleic acidmolecule of claim 7 wherein said nucleic acid molecule comprises thepolynucleotide sequence described in SEQ ID NO:
 3. 9. A recombinantexpression vector comprising the nucleic acid molecule of claim
 7. 10.The recombinant expression vector of claim 9 wherein said nucleic acidmolecule comprises the polynucleotide sequence described in SEQ ID NO:3.
 11. A host cell comprising the recombinant expression vector of claim9.
 12. A gene delivery system comprising: (a) the nucleic acid moleculedescribed in SEQ ID NO:1; or (b) the nucleic acid molecule comprisingthe nucleic acid sequence of SEQ ID NO:3.
 13. The gene delivery systemof claim 12, wherein said isolated nucleic acid molecule is present in acationic lipid complex.
 14. The gene delivery system of claim 12,wherein said isolated nucleic acid molecule is present in a viralvector.
 15. An antibody having immunospecificity for the polypeptidesequence of SEQ ID NO:2.